In the gel electrophoretic resolution of oligonucleotides synthesized by a DNA or RNA polymerase-catalyzed reaction, artifacts occur when local sequence permits areas of secondary structure to occur in single-stranded nucleic acids, even under nominally denaturing conditions.
Resolution of DNA sequencing reaction mixtures by electrophoresis through denaturing polyacrylamide gels is often locally impaired by irregularities in the spacing of the electrophoretic bands. This phenomenon usually occurs as "band compression", a reduction in the spacing between consecutive bands, which may render their correct reading impossible. Band compression in sequencing gels occurs when the corresponding local nucleotide sequence contains an inverted repeat, and it is thought to arise from formation of hairpin structures in the single-stranded DNA fragments, despite the presence of 7M urea in the polyacrylamide gel. To alleviate this problem, the dGTP in the synthesis mixtures has been substituted by 7-deaza-dGTP (Barr PJ et al. (1986) BioTechniques.4:428-432; Mizusawa S et al. (1986) Nucleic Acids Res. 14:1319-1324) or by dITP. However, the use of 7-deaza-dGTP does not lead to complete resolution of band compressions caused by extended inverted repeats (which give rise to particularly stable hairpin loops) , and use of dITP often leads to false stops at G-sites.
In applications of a polymerase chain reaction, hairpin structures formed by nearby G:C-rich tracts of a single-stranded template can reduce the fidelity of copying, resulting in amplification of incomplete and of incorrect sequences when compared to the original template.
Replacement of cytosines by N.sup.4 -methylcytosines (N.sup.4 -methylC) in polynucleotides has been shown to lower the stability of the polynucleotide complexes. The reduced stability of the G.N.sup.4 -methylC pair, compared to the G.C pair, has been documented in short self-complementary oligonucleotides (Fazakerley GV et al. (1987) Nucleic Acids Res. 15:2191-2200; Butkus V et al. (1987) Nucleic Acids Res. 15:8467-8478). When those data are compared with the results obtained in a comparison of the melting temperatures of poly(G).poly(C) and poly(7-deazaG).poly(C) (Seela F et al. (1982) Biochemistry 21:4338-4343), the destabilization imparted to the G.C base pair by N.sup.4 -methylation of the cytosine appears to be much greater than the destabilization of the G.C pair caused by substituting 7-deazaguanine for guanine.
N.sup.4 -methyl-2'-deoxycytidine has been synthesized previously and incorporated into chemically synthesized oligonucleotides (Butkus et al., supra) . Conversion of C residues in RNA to N.sup.4 -methylC has been reported by Draper DE (1984) Nucleic Acids Res. 12:989-1002.
The duplex poly(7-deazaG).poly(C) shows only slightly lower thermal stability, compared to poly(G).poly(C), with T.sub.m values of 74.degree. C. and 75.degree. C., respectively, measured in 0.2M sodium EDTA at pH 5.3 (Seela et al., supra). In contrast to this moderate effect, the destabilization of the G.C pair achieved by the substitution of N.sup.4 -methylcytosine for cytosine is more substantial: for the duplex formed from a fully self-complementary dodecadeoxyribonucleotide, conversion of only two base pairs from G.C to G.N.sup.4 -methylC resulted in a change of T.sub.m from 60.degree. C. to 55.degree. C., measured in 0.1M NaCl, 0.2 mM EDTA, at 0.24 mM total nucleotide concentration (Butkus et al., supra), and for the duplex formed from the hexamer d-CGCGCG, change of the two central base pairs from G.C to G.N.sup.4 -methylC is reported to depress the T.sub.m by about 19.degree. C., measured in D.sub.2 O solution containing 150 mM NaCl, 10 mM phosphate (pH 7.4), and 0.2 mM EDTA at 48 mM total nucleotide concentration (Fazakerley et al., supra). At the polynucleotide level, complete replacement of cytosines in poly(I).poly(C) by N.sup.4 -methylcytosines lowers the T.sub.m by at least 50.degree. C., and 39% replacement of cytosines by N.sup.4 -methylcytosines results in a T.sub.m depression of 15.degree. C. (Brimacombe RLC & Reese CB (1966) J. Mol. Biol. 18:529-540).